Synthesis of N-(3-Acyloxyacyl)glycines, Small Molecules with Potential Role in Gut Microbiome-Endocannabinoidome Communication

The synthesis of some N-(3-acyloxyacyl)glycines, an interesting class of bioactive gut microbiota metabolites, is described. This procedure involves seven reaction steps using the commercially available Meldrum’s acid to obtain highly pure products, in normal or deuterated form. The key point of the synthetic strategy was the use of commendamide t-butyl ester as a synthetic intermediate, a choice that allowed the removal of the protecting group at the end of the synthetic procedure without degrading of the other ester bond present in the molecule. The developed synthetic sequence is particularly simple, uses readily available reagents and involves a limited number of purifications by chromatographic column, with a reduction in the volume of solvent and energy used.


Introduction
N- (3-acyloxyacyl)glycines are an important class of bioactive products of bacterial origin.Molecules of this type were isolated by Morishita et al. from a marine bacterium, Cytophaga sp.SANK 71996, [1] and, through a refined work of structural elucidation, it was possible to identify them.The most active molecules among those isolated had branchedchain acyl groups (1a, 1b and 2, Figure 1) and proved to be highly selective inhibitors for N-type calcium channels (localized primarily in the nervous system and involved in the release of neurotransmitters) vs. L-type channels.

Introduction
N- (3-acyloxyacyl)glycines are an important class of bioactive products of bacterial origin.Molecules of this type were isolated by Morishita et al. from a marine bacterium, Cytophaga sp.SANK 71996, [1] and, through a refined work of structural elucidation, it was possible to identify them.The most active molecules among those isolated had branched-chain acyl groups (1a, 1b and 2, Figure 1) and proved to be highly selective inhibitors for N-type calcium channels (localized primarily in the nervous system and involved in the release of neurotransmitters) vs. L-type channels.Natural products with a similar structure, known as Topostins B, have also been isolated from a culture broth of Flexibacter topostinus sp.Nov. [2] and in this case, after the 2 of 9 structural identification, their bioactivity as inhibitors of mammalian DNA topoisomerase I was also highlighted (products 1b and 3, Figure 1) [3].It should also be noted that some of these molecules (for example Topostin B567) have the same structure as the metabolites produced by the marine bacterium Cytophaga sp.SANK 71996 (in this case, 1b).Interestingly, N-(3-acyloxyacyl)glycines bear a chemical resemblance to mammalian linear fatty acid esters of hydroxyl fatty acids (FAHFAs), which also contain an ester bond between the two chains (described as the "estolide" bond), although it is from the C-5 of the lower chain, and possess key biological activities in the regulation of diabetes and inflammation [4].
Against this background, the recent discovery that even bacteria belonging to the mammalian gut microbiome (Bacteroides thetaiotaomicron) are able to produce N-(3-acyloxyacyl)gl ycines appears particularly noteworthy [5,6].In particular, Lynch et al. identified a gene from Bacteroides, glsB (BT_3459), which encodes an N-acyltransferase required for the initial production of N-(3-hydroxyacyl)glycines such as commendamide [7,8] (a gut microbiotaderived bioactive metabolite with a very similar structure to long-chain N-acyl amino acids belonging to the endocannabinoidome [9]), and another immediately close gene, glsA (BT_3458), which encodes an O-acyltransferase, required for the subsequent esterification of the hydroxy group with endogenous or dietary fatty acids.
Differently from what has been reported for other types of bacteria, the biosynthesis of N-(3-acyloxyacyl)glycines with linear acyl chains (such as N-[3-(palmitoyloxy)palmitoyl]gly cine, i.e., the derivative of commendamide acylated with palmitic acid [5]), seems possible in the case of the mammalian gut microbiota and this different situation might be explained considering that, in this case, linear long-chain fatty acids are widely available in the host (as endogenous or dietary precursors), and, therefore, they can be used as synthons for the biosynthesis of N-(3-acyloxyacyl)glycines.
Some of these N-(3-acyloxyacyl)glycines seem to play a fundamental role in the adaptation processes of Bacteroides to environmental stress but also in the colonization process of the mammalian gut by beneficial bacteria.The identities of most of these molecules were inferred by tandem MS/MS analysis although, by using only this technique, sometimes it was not possible to demonstrate the presence of straight-or branched-chain acyl groups in the molecules with certainty [5].
The discovery that gut microbiota are able to biosynthesize N-(3-acyloxyacyl)glycines is also extremely important because, in this case, potentially bioactive molecules are immediately bioavailable in the host, considering that gut microbiota live in symbiosis with the latter and therefore these metabolites could have a direct impact on human physiology.In this regard, it is important to note that varied experimental evidence highlights the close interaction between the gut microbiome and the endocannabinoidome, with microbial metabolites that can interact with molecular targets of the endocannabinoidome and vice versa (the gut microbiome-endocannabinoidome axis), [10] which is also thanks to the structural similarities that often exist between microbial metabolites and endogenous signal molecules, as highlighted above.All this considered, the identification of new molecules that can act as chemical signals in host-microbe communication, but also the understanding of the molecular mechanisms underlying this communication, is fundamental and, in this context, the N-(3-acyloxyacyl)glycines seem to be able to play an important role.
Organic synthesis [11] is fundamental for the chemical and biomolecular study of the microbiome, not only for the production of greater amounts of natural products with the aim of extending their pharmacological screening and identifying new molecular mechanisms of interaction with the host, but also to achieve unequivocal structural identification of new metabolites (thanks to the synthesis of pure standards that can be compared to products extracted from biological matrices).Therefore, the elaboration of simple and effective synthetic protocols that, if necessary, can also be used for the synthesis of products in deuterated form (useful in drug discovery [12] and for the development of LC-MS quantitative analysis methods), is very important.In fact, in the development of new LC-MS methods for the quantification of analytes in biological matrices, the use of internal standards [13,14] is fundamental and, in this context, a stable isotope-labeled form of the molecule to be quantified (such as the deuterated analogue, which is not prone to exchange/loss of the isotopic label) is the best internal standard, because it has chemical-physical properties very similar to the analyte (the same extraction recovery, ionization response in ESI-MS, and chromatographic retention time) so it can be co-eluted with the analyte.However, their molecular masses are well distinguishable.On the other hand, the structural properties of N-(3-acyloxyacyl)glycines and the bioactivities highlighted for many molecules belonging to this class, as well as the presence of a scaffold found also in other bioactive products (WB-3559 A, B, C and D; Flavolipin; FAHFAs; etc.), [4,15,16] make them an interesting target for chemical synthesis.
In this work, an easy and versatile methodology for the synthesis of some N-(3acyloxyacyl)glycines was developed, starting from the synthetic strategy previously reported for commendamide and its analogues [17].We describe a synthetic sequence characterized by the use of simple workups, readily available reagents and minimal volumes of organic solvents.

Results and Discussion
The synthesis of commendamide and its analogues was recently carried out in six steps, starting with the acylation of Meldrum's acid 4 with a suitable acyl chloride (Scheme 1) [17].Interestingly, none of the steps involved the use of halogenated solvents and most of the reactions proceeded with high yields, so it was often possible to work directly on the unpurified reaction crudes.In fact, in the multi-step synthesis reported in Scheme 1, only two chromatographic column purifications are required with considerable simplification of the experimental procedures and reduction in the volume of solvent and energy used.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 9 quantitative analysis methods), is very important.In fact, in the development of new LC-MS methods for the quantification of analytes in biological matrices, the use of internal standards [13,14] is fundamental and, in this context, a stable isotope-labeled form of the molecule to be quantified (such as the deuterated analogue, which is not prone to exchange/loss of the isotopic label) is the best internal standard, because it has chemicalphysical properties very similar to the analyte (the same extraction recovery, ionization response in ESI-MS, and chromatographic retention time) so it can be co-eluted with the analyte.However, their molecular masses are well distinguishable.On the other hand, the structural properties of N-(3-acyloxyacyl)glycines and the bioactivities highlighted for many molecules belonging to this class, as well as the presence of a scaffold found also in other bioactive products (WB-3559 A, B, C and D; Flavolipin; FAHFAs; etc.), [4,15,16] make them an interesting target for chemical synthesis.
In this work, an easy and versatile methodology for the synthesis of some N-(3acyloxyacyl)glycines was developed, starting from the synthetic strategy previously reported for commendamide and its analogues [17].We describe a synthetic sequence characterized by the use of simple workups, readily available reagents and minimal volumes of organic solvents.

Results and Discussion
The synthesis of commendamide and its analogues was recently carried out in six steps, starting with the acylation of Meldrum's acid 4 with a suitable acyl chloride (Scheme 1) [17].Interestingly, none of the steps involved the use of halogenated solvents and most of the reactions proceeded with high yields, so it was often possible to work directly on the unpurified reaction crudes.In fact, in the multi-step synthesis reported in Scheme 1, only two chromatographic column purifications are required with considerable simplification of the experimental procedures and reduction in the volume of solvent and energy used.Previous work Synthesis of normal and deuterated commendamide 11 [17].
With the aim of synthesizing some N- (3-acyloxyacyl)glycines between commendamide and bioavailable long-chain fatty acids (endogenous or dietary LCFA), the synthetic strategy reported in Scheme 1 was applied to obtain the intermediates 10; then, the products 10a and 10b were submitted to an esterification reaction with oleoyl chloride 12 before the subsequent basic hydrolysis.
Unfortunately, basic hydrolysis led to the complete degradation of the intermediate 13a (also at low temperature), with consequent formation of commendamide 11a and oleic acid, due to the hydrolysis of both ester bonds present in the molecule.Therefore, it was not possible to obtain the product 14a, while the deuterated product 14b was isolated only in a very small amount (Scheme 2).An alternative method for the selective hydrolysis of Scheme 1. Synthesis of normal and deuterated commendamide 11 [17].
With the aim of synthesizing some N- (3-acyloxyacyl)glycines between commendamide and bioavailable long-chain fatty acids (endogenous or dietary LCFA), the synthetic strategy reported in Scheme 1 was applied to obtain the intermediates 10; then, the products 10a and 10b were submitted to an esterification reaction with oleoyl chloride 12 before the subsequent basic hydrolysis.
Unfortunately, basic hydrolysis led to the complete degradation of the intermediate 13a (also at low temperature), with consequent formation of commendamide 11a and oleic acid, due to the hydrolysis of both ester bonds present in the molecule.Therefore, it was not possible to obtain the product 14a, while the deuterated product 14b was isolated only in a very small amount (Scheme 2).An alternative method for the selective hydrolysis of methyl esters with LiCl-DMF [18] was ineffective on the substrates 13.Thus, a modified synthetic sequence was used in order to obtain the products 14.Indeed, 3-hydroxypalmitic acid 8, synthesized as shown in Scheme 1, was subjected to a coupling reaction with glycine t-butyl ester 15 (Scheme 3), instead of glycine methyl ester 9, as reported also by Morishita et al. for the synthesis of similar products [1].
methyl esters with LiCl-DMF [18] was ineffective on the substrates 13.Thus, a modified synthetic sequence was used in order to obtain the products 14.Indeed, 3-hydroxypalmitic acid 8, synthesized as shown in Scheme 1, was subjected to a coupling reaction with glycine t-butyl ester 15 (Scheme 3), instead of glycine methyl ester 9, as reported also by Morishita et al. for the synthesis of similar products [1].methyl esters with LiCl-DMF [18] was ineffective on the substrates 13.Thus, a modified synthetic sequence was used in order to obtain the products 14.Indeed, 3-hydroxypalmitic acid 8, synthesized as shown in Scheme 1, was subjected to a coupling reaction with glycine t-butyl ester 15 (Scheme 3), instead of glycine methyl ester 9, as reported also by Morishita et al. for the synthesis of similar products [1].The corresponding commendamide t-butyl ester 16 was then esterified with three different long-chain fatty acid chlorides (oleoyl-chloride 12, palmitoyl-chloride 17 and its deuterated analogue 18) with good yields, and the obtained products (19, 20 and 21) were finally deprotected by treatment with trifluoroacetic acid (TFA) at room temperature to yield N-(3-acyloxyacyl)glycines 14.In fact, TFA was able to selectively remove t-butyl esters at rt, without hydrolyzing the other ester bond present in the molecule.Thanks to this modified procedure (Scheme 3), the products 14 were obtained with a total yield of up to 40% starting from Meldrum's acid 4 (seven steps).

General
All reagents and solvents were Merck-SigmaAldrich and used as received.The reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel plates (0.25 mm) and visualized by UV light at 254 nm and cerium sulfate reagent. 1 H NMR and 13 C NMR spectra were recorded on a Bruker DRX 600 spectrometer equipped with a three-channel inverse TCI CryoProbe (Bruker, Milan, Italy) at room temperature in CDCl 3 .Chemical shifts are reported in ppm; multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad).Coupling constants (J) are reported in Hz.IR measurements were conducted on KBr pills with Vertex 70 Bruker spectrophotometer (Bruker, Milan, Italy) and maximum absorptions are reported in wavelength (cm −1 ).Highresolution mass spectra (HRMS) were acquired using an OrbiTrap high-resolution mass spectrometer (Q Exactive) equipped with the heated electrospray ionization probe HESI-II (Thermo Fisher Scientific, Bremen, Germany) operating in both positive and negative ion modes.Yields are given for isolated products showing one spot on a TLC plate and no impurities detectable in the NMR spectrum.The synthetic procedures and spectroscopic characterizations of products 6, 7, 8, 10a and 10b are reported in the literature [17]. 1 H and 13 C NMR spectra of products 13a, 13b, 16, 19, 20, 21, 14a, 14b, 14c and 14d are reported in Supplementary Materials.